Regulation of TLR trafficking for proper self versus non-self discrimination The strategy of sensing nucleic acids by the innate immune system enables broad recognition of pathogens but exposes the host to the risk of autoimmunity. Recognition of self RNA and DNA by members of the Toll-like receptor (TLR) family can drive pathology in autoimmune diseases such as lupus. How TLRs are regulated to permit recognition of pathogens while avoiding self recognition remains a key unanswered question. This proposal addresses this gap in our understanding by focusing on a critical TLR regulatory protein called Unc93b1. Previous work by our group and by other groups has shown that Unc93b1 controls the trafficking and localization of endosomal TLRs. We recently performed a saturating mutagenesis screen of Unc93b1 to systematically dissect Unc93b1 function, and the results of this screen have transformed our view of Unc93b1's role in TLR regulation. Our preliminary results support three new concepts: (1) Unc93b1 is a multi- functional adaptor that has distinct functional domains that specifically regulate individual nucleic acid-sensing TLRs; (2) Unc93b1 is more than a trafficking chaperone, but appears to be critical for regulation of TLR signaling itself; (3) Unc93b1 not only positively regulates TLRs, but also appears to play an important role in negative regulation of TLR signaling. We investigate these concepts over three Aims. Aim 1 seeks to identify the molecular mechanisms by which Unc93b1 positively regulates trafficking and function of individual TLRs. Aims 2 and 3 will explore how Unc93b1 negatively regulates TLR7 and TLR3 responses to endogenous RNAs. Altogether, the work in this proposal will discover new pathways that regulate TLR function and will greatly improve our understanding of how TLRs balance recognition of microbial versus self nucleic acids.